The rapid explosion in genetic knowledge coming from high-throughput genome sequencing of patient DNA is generating unprecedented numbers of novel alleles correlated with both rare and common diseases. Many of these newly discovered alleles are in genes whose function is not known. In order to build models of these genetic diseases, geneticists generate knockout mutations in the gene and study the resulting phenotype. Phenotypic analysis can provide confirmation that a particular gene can contribute to the disease state and provide a tool to better understand the disease. However, a better understanding of gene function requires identification of the genetic pathways that the disease gene is required for. To identify these genetic pathways many approaches can be taken, but unbiased screening approaches have the advantage that they are model independent and can be applied to many different disease-associated genes in an analytical pipeline. Classically, genetic suppressor/enhancer screens have been used to identify pathways and genetic interactions, however this approach is time consuming and laborious. More recently, chemical interaction screens have been developed in which well-characterized, pharmacologically-active small molecule libraries are screened against the mutants. This approach is amenable to high throughput pipelines. Interactors are those small molecules that ameliorate or specifically exacerbate the phenotype. Since the protein targets of these libraries are well characterized, the pathway can be identified from these screening techniques. These results can then be confirmed through genetic interaction with the drug target genes. Our laboratories have developed new genome engineering technologies that allow for the rapid generation of mutations in the zebrafish genome. We will utilize this crispr-directed mutagenesis system to generate mutations in each of the zebrafish orthologs of the genes identified by the NIH Undiagnosed Disease Program. To begin to characterize these largely uncharacterized genes, the expression pattern of the genes will be analyzed during the first 5 days of life by in situ hybridization. The generated mutants will be analyzed for anatomical/developmental defects as well as functional differences in behavior and physiology. Many of the UDP-identified alleles are associated with neurological disease and particular attention for those genes will be placed on midbrain and hindbrain neuroanatomy, optokinetic response, motility, autonomic regulation of the heart, and ss cell mass changes as appropriate for the disease phenotype. The phenotypes most amenable to small molecule library screening will then be tested for interaction against a small molecule library of pharmacologically active and well characterized compounds. This compound screen will serve two purposes: First, the screen will identify compounds that interact with the allele which will assist in gene function model building. Second, the compound screen may identify pharmaceuticals that may be of direct clinical use to the patient.